Cloning and expression of the pyridoxal 5′-phosphate-dependent aspartate racemase gene from the bivalve mollusk Scapharca broughtonii and characterization of the recombinant enzyme
Cloning and expression of the pyridoxal 5′-phosphate-dependent aspartate racemase gene from the bivalve mollusk Scapharca broughtonii and characterization of the recombinant enzyme
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DOI：
10.1093/jb/mvj028
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发表时间：
2006-02-01
影响因子：
2.7
通讯作者：
Yamada, RH
中科院分区：
文献类型：
--
作者：
Abe, K;Takahashi, S;Yamada, RH
D-天冬氨酸在布氏舟蛾的组织中以高浓度存在，其产生依赖于天冬氨酸消旋酶。这种酶是第一种从动物组织中纯化的天冬氨酸消旋酶，与迄今为止表征的微生物天冬氨酸消旋酶相比，其独特之处在于其吡哆醛5 '-磷酸（PLP）依赖性。在AMP存在下，酶活性显著增加，在ATP存在下，酶活性降低。为了进一步分析天冬氨酸消旋酶的结构与功能的关系，我们克隆了天冬氨酸消旋酶的cDNA，并在大肠杆菌中表达了重组天冬氨酸消旋酶。该cDNA包含一个1,017 bp的开放阅读框，编码338个氨基酸，推导的氨基酸序列含有PLP结合基序。该序列与哺乳动物丝氨酸消旋酶具有最高的同一性（43-44%），其次主要是苏氨酸消旋酶。这些关系完全支持的酶的系统发育分析。在大肠杆菌提取物中发现的活性重组天冬氨酸消旋酶约占细菌总蛋白的10%，并经纯化以显示与天然酶基本相同的物理化学和催化性质。此外，该酶显示出对L-苏型-3-羟基天冬氨酸的脱氢酶活性，类似于从D-和L-丝氨酸产生丙酮酸的哺乳动物丝氨酸消旋酶。
D-Aspartate is present at high concentrations in the tissues of Scapharca broughtonii, and its production depends on aspartate racemase. This enzyme is the first aspartate racemase purified from animal tissues and unique in its pyridoxal 5'-phosphate (PLP)-dependence in contrast to microbial aspartate racemases thus far characterized. The enzyme activity is markedly increased in the presence of AMP and decreased in the presence of ATP. To analyze the structure-function relationship of the enzyme further, we cloned the cDNA of aspartate racemase, and then purified and characterized the recombinant enzyme expressed in Escherichia coli. The cDNA included an open reading frame of 1,017 bp encoding a protein of 338 amino acids, and the deduced amino acid sequence contained a PLP-binding motif. The sequence exhibits the highest identity (43-44%) to mammalian serine racemase, followed mainly by threonine dehydratase. These relationships are fully supported by phylogenetic analyses of the enzymes. The active recombinant aspartate racemase found in the Escherichia coli extract represented about 10% of total bacterial protein and was purified to display essentially identical physicochemical and catalytic properties with those of the native enzyme. In addition, the enzyme showed a dehydratase activity toward L-threo-3-hydroxyaspartate, similar to the mammalian serine racemase that produces pyruvate from D- and L-serine.